Evidence exists for an essential role of the 12-lipoxygenase (12-LO) pathway in Ang II-stimulated aldosterone secretion. However, the identity of 12-LO metabolites, as well as the mechanism and extent by which these metabolites contribute to Ang II-stimulation of aldosterone secretion remains unknown. The long-term goal of these studies is to understand the role of 12-LO in the regulation of aldosterone secretion. The objective of this proposal is to identify the 12-LO metabolites that contribute to Ang II-stimulated aldosterone secretion and to characterize the receptor that mediates the action of these active metabolites. The central hypothesis is that arachidonic acid (AA)-derived hydroxyeicosatetraenoic acids (HETEs) and/or adrenic acid (AdA)-derived hydroxydocosatetraenoic acids (HDTEs) mediate Ang II-stimulated aldosterone secretion by activation of a guanine nucleotide binding protein (G-protein) coupled receptor (GPCR). This hypothesis has been formulated on the basis of preliminary data produced in the applicant's laboratory. The rationale for the proposed research is that identification of the 12-LO metabolites and their receptor that mediates aldosterone secretion would provide a novel pharmacological target for the remediation of circulating aldosterone levels. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify the 12-LO metabolites that contribute to aldosterone secretion; and 2) Characterize the receptor that mediates the activity of 12-LO metabolites in ZG cells. Under the first aim, 12-LO AA and AdA metabolites that increase following these steroidogenic stimuli will be identified by liquid chromatography/mass spectrometry (LC/MS), and then these metabolites will be tested for steroidogenic activity. Under the second aim, the ability and potency of these 12-LO AA and AdA metabolites to activate a GPCR on ZG membranes will be tested by GTP?[35S] binding, as well as their ability to increase cAMP and intracellular calcium. Additionally, the type of Ga subunit that is activated by these metabolites will also be determined. Furthermore, analogs of 12-HETE and 14- HDTE will be used to determine the functional groups necessary for both receptor activation and calcium signaling. The approach is innovative, because it will develop specific LC/MS methods to measure adrenal HETEs and HDTEs and define the role of both AA and AdA metabolites in aldosterone production. The proposed research is significant, because the characterization of a 12(S)-HETE and/or 14(S)-HDTE receptor is an essential step towards understanding the critical role of 12-LO metabolites in the regulation of aldosterone secretion. Ultimately, characterization of this novel GPCR will provide an innovative pharmacological target to not only ameliorate cardiovascular disease associated with hyperaldosteronism, but potentially many other diseases in which 12-LO metabolites have been implicated including inflammatory, hyperproliferative, and hyperplastic diseases such as atherosclerosis, diabetes, Parkinson's disease, Alzheimer's disease, and cancer.